chp 9 cell bio

• Primary cell cultures & cell strains have a finite life span
• tissues fragments are treated w a combination of protease (trypsin) & divalent cation chelator (EDTA) that depletes the medium of free Ca2+
• A subculture is new cell or microbiological culture made by transferring some or all cells from a previous culture to a fresh growth medium. this action is called subculturing or passaging the cells
• Most of adherent cells divide a finite number of time then cease growing ( human fetal fibroblast that divide about 50 time before cease growth )
• A fibroblast is a type of cell that synthesize the extracellular matric & collagen, most common cell of connective tissue in animals
• transformed cells can grow indefinitely in culture
• Biologist often want to maintain c culture for many more than 50 doublings. Such prolonged growth is exhibited by c derived from some tumor
• Rare cell in a population of primary cell may undergo spontaneous oncogenic mutation leading to oncogenic transformation
• A culture cell with an indefinite life span is considered immortal and is called a cell line
• Hybrid cells called Hybridomas produces abundant Monoclonal antibodies
• Phase-contrast & differential-interference- contrast (DIC) microscopy visualize unstained living cells
• Imaging subcellular details often requires that the samples be fixed, sectioned and stained
• Hematoxylin : dark blue / violet stain that is basic/positive. It binds to basophilic substances (such as DNA/RNA- which r acidic & negatively charged
• Eosin : red/pink stain that is acidic/negatively. Binds to acidophilic substance such as positively charged amino acid side chains (ex: lysine, arginine)
• DNA in the nucleus r both acidic bc phosphate backbones of nucleic acids r negatively charged
• Most proteins in cytoplasm r basic because they r positively charged dye to arginine & lysine amino acid residues
• Immunofluorescence Microscopy can detect specific protein in fixed cells . To localize a specific protein by immunofluorescence microscopy, a tissue section or sample of c has to be mechanically fixed and made permeable to antibody. GLUT2, a glucose transport protein was localized by indirect immunofluorescence microscopy
• Deconvolution and confocal microscopy enhance visualization of 3D fluorescent objects.
• Single mol or Structures can be Imaged after a negative stain or metal shadowing : transmission electron microscope (TEM)

  • TEM r used mainly to study the internal structure of cells.

  Ex: rotaviruses
• Immunoelectron Microscopy localized Proteins at the Ultrastructural level

  Ex: catalase located only in peroxisomes
• Scanning electron Microscopy of Metal-Coated specimens reveals surface features

  • Scanning electron microscopes (SEM) focus on bean of e- onto the surface of a specimen providing 3D look.

  Ex: epithelium in lumen
• Homogenization of cell in a blender to break them down
• Result in a homogenate structure that is centrifuged. The supernatant (liquid) is poured into another tube and centrifuged at a higher speed for a longer time.
• Organelles-Specific Antibodies are useful in preparing highly purified organelles
• Interaction of protein ab bound to clathrin-coated vesicles links the vesicles to the bacterial cells. The vesicle-bacteria complexes can then be recovered by low-speed centrifugation.
• Drugs are commonly used in cell biology